Reaction sensing in living cells

ABSTRACT

Chemical reactions occurring within a living cell are measured in a manner that does not affect the viability of the cell or the reaction under study. In one embodiment, one or more sensors are introduced into the cell and/or covalently associated with the exterior cell membrane. The sensor(s) emit an observable signal indicating a value of a parameter associated with the chemical reaction, e.g., the concentration of a reaction product. Because cell viability is not compromised, the cell may be stimulated (e.g., by subjection to an agonist or antagonist, a pathogen, a pharmaceutical compound, or a potential toxin) so as to affect the reaction under study.

CROSS-REFERENCE TO RELATED CASES

This application claims the benefit of U.S. Provisional Patent Application No. 60/718,255, entitled “Optical Biosensor Array and Related Systems, Methods and Devices,” filed on Sep. 16, 2005, the entire disclosure of which is hereby incorporated by reference as if set forth herein in its entirety.

FIELD OF THE INVENTION

The invention relates to monitoring chemical reactions occurring within living cells, and more particularly to methods and apparatus for measuring such reactions without compromising the viability of the cells.

BACKGROUND OF THE INVENTION

Numerous techniques exist for monitoring the extent and time profile of a chemical reaction of interest occurring within a living cell. Many such techniques are indirect, involving an assay for reaction products outside the cell that reflect the progress of reaction(s) taking place in the cytoplasm. A disadvantage of such assays is their vulnerability to influence by the spurious presence of reaction products in the cell's environment that do not, in fact, emanate from the cell.

Intracellular techniques have also been developed; these include, for example, genetically engineering cells to fluoresce in the presence of a compound of interest, radioactive binding assays, and colorimetric assays. These techniques, however, require potentially destructive modification or invasion of the cell.

Intracellular and extracellular assays are often employed to track enzymatic activity by measuring the amount of reaction product through the use of coupling. Enzymatic coupling is a complicated approach that links a non-quantifiable enzymatic reaction of interest with an optically measurable (by means of a spectrophotometer) enzymatic reaction that will interact with the products of the enzymatic reaction of interest. Once again, conventional approaches suffer from a number of deficiencies. For example, coupled reactions necessarily involve at least one chemical reaction unrelated (“coupled”) to the enzymatic activity of interest, imparting potential sources of error. Enzymatic reactions require carefully controlled reaction conditions (appropriate temperatures, pHs, salt concentrations, etc.) and, as a result, monitoring efforts can be affected by rapid environmental changes or degradation of reagents, e.g., components of the enzymatic reactions (especially the enzyme itself).

Accordingly, there is a need for apparatus and methods that quickly, directly and accurately measure a product of interest.

SUMMARY OF THE INVENTION

The present invention addresses the shortcomings of the prior art by measuring chemical reactions occurring within a living cell in a manner that does not significantly affect the viability of the cell or the reaction under study. By “significantly” is meant, with respect to individual cells, that these are not killed; with respect to a population of cells, that at least the majority remains viable; and with respect to a reaction, that the variable under study is not affected beyond a predetermined threshold of precision. For example, if it is necessary to monitor the concentration of an ion to within 0.1 mole/L, the reaction is not significantly affected if the ion concentration is not shifted more than ±0.05 mole/L.

In one embodiment, one or more sensors are introduced into the cell and/or covalently associated with the exterior cell membrane. The sensor(s) produce an observable signal indicating a value of a parameter associated with the chemical reaction, e.g., the concentration of a reaction product. Because cell viability is not compromised, the cell may be stimulated (e.g., by subjection to an agonist or antagonist, a pathogen, a pharmaceutical compound, or a potential toxin) so as to affect the reaction under study. By “observable” is meant visible or measurable using instrumentation.

In some embodiments, the sensor is used to monitor the activity of an ion channel through the cell membrane. In such cases, the sensor may be introduced within the cell, may be attached to the exterior cell membrane, or both. For example, the external sensor may be attached to the cell membrane proximate to an ion channel of the cell, e.g., via an antibody specific to the ion channel (which typically comprises a protein). The sensor may be an ion sensor, and sensors internal and external to the cell may monitor different ions.

In some embodiments, the sensor comprises an ion-selective optode. The reaction may produce an ionic product that is sequestered by the optode, or instead, the reaction may produce a non-ionic product that is ionized to facilitate monitoring using the optode. In this way, the invention can be used in numerous pharmaceutical applications involving small molecules.

In some embodiments, the optode comprises an ion-selective ionophore, a source of triggering ions, and a signaling agent responsive to the triggering ions. Typically the ionophore is selective for the ionic reaction product, and upon binding of a reaction-product ion, a triggering ion is released to interact with the signaling agent and thereby produce the signal. The strength of the signal may be indicative of the concentration of the triggering ion, which itself reflects the concentration of the ionic reaction product. Different sensors, each responsive to different ions and producing differentiable signals, can be used depending on the reaction(s) being monitored. Furthermore, the signal may be produced directly by the triggering ions, or may be produced indirectly (e.g., the triggering ions may initiate a reaction cascade that results in an observable signal).

In preferred embodiments, the signal is optical in nature, e.g., involving fluorescence or absorbence. The optical signal may be monitored using, for example, a spectrometer, a fluorometer, or a detector for measuring absorbance. But the signal may alternatively be non-visible or may involve other forms of electromagnetic radiation.

The foregoing and other features and advantages of the present invention will be made more apparent from the description, drawings, and claims that follow.

BRIEF DESCRIPTION OF DRAWINGS

The advantages of the invention may be better understood by referring to the following drawings taken in conjunction with the accompanying description in which:

FIG. 1 is a schematic diagram of a cell assay system according to an illustrative embodiment of the invention.

FIGS. 2A-2D are cross-sections of various optical ion sensor arrangements suitable for use in various implementations of the cell assay system of FIG. 1.

FIG. 3 presents another embodiment of a microfluidic device in accord with the present invention.

FIG. 4 depicts the embodiment of FIG. 5 with fluorescence from the channels imaged.

FIG. 5 presents a hypothetical response of yeast cells to a stimulus (Haloperidol).

FIG. 6 presents data from an exemplary study using a sensor in accord with the present invention to monitor the reaction of yeast cells to Haloperidol. Sodium is shown on the left and potassium on the right.

FIG. 7 illustrates an embodiment of the present invention used as an ammonia sensor.

FIG. 8 depicts the calibration curve for the embodiment of FIG. 7.

FIG. 9 presents the data from real-time monitoring of Tgase reactions using sensors in accord with one embodiment of the present invention, using 12.5 mM substrate (Z-Gln-Gly) and 1.00, 0.20, and 0.02 units/ml Transglutaminase at 30° C. Each curve represents the average reading from 5-8 reactions.

FIG. 10 presents the data from real-time monitoring of TGase reactions using sensors in accord with another embodiment of the present invention, having various substrate (Z-Gln-Gly) concentrations and 0.4 units/ml Transglutaminase at 30° C. Each curve represents the average reading from 3 reactions.

In the drawings, like reference characters generally refer to corresponding parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed on the principles and concepts of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In brief overview, embodiments of the present invention provide systems, methods and devices for measuring cellular biomarkers. In exemplary embodiments, optically active polymer sensors are placed inside or outside a cell. Changes in fluorescence or absorbance indicate the ion concentrations and fluxes from the cell. The overall change in fluorescence or absorbance in response to a compound of interest provides a “fingerprint” (e.g., a spectrum) for that compound.

FIG. 1 is a schematic diagram of a cell assay system 100 according to an illustrative embodiment of the invention. The cell assay system 100 includes an optical ion sensor support 102, a biological sample holder 104, an excitation light source 106, a light sensor 108, and a computing device 111.

The optical ion sensor support 102 supports an optical ion sensor 112 for positioning in the biological sample holder 104. In various implementations, the optical ion sensor 112 is adhered to the optical ion sensor support 102 by deposition in a solution of optical ion sensor matrices dissolved or dispersed in a solvent, such as in a polar organic solvent like tetrahydrofuran (THF). In such implementations, the sensor support 102 is preferably formed from a material resistant to the solvent. Materials resistant to THF include, without limitation, 304 stainless steel; 316 stainless steel; acetal polymer (marketed as DELRIN by E. I. du Pont de Nemours and Company); bronze; carbon graphite; carbon steel; ceramic Al₂O₃; a perfluoroelastomer compound, such as CHEMRAZ marketed by Greene, Tweed; epoxy; HOSTELRY Calloy (marketed by Haynes International, Inc.); KALES elastomer (marketed by DuPont Performance Elastomers); polychlorotrifluoroethylene; NYLON (marketed by E. I. du Pont de Nemours and Company); polyetherether ketone (PEEK); polyphenylene sulfide; and PTFE.

The optical ion sensor 112 includes a film having a suspension of optical ion sensor matrices. The optical ion sensor matrices, in general, include an ionophore, an additive, and a chromionophore suspended in a polymer phase, for example, of polyvinyl chloride (PVC). The polymer phase also includes a plasticizer such as dioctyl sebacate (DOS). An ionophore is a substance that allows targeted ions to move across or into a membrane. Preferably the ionophore is selected to be lipid-soluble. In addition, the ionophore is preferably an electrically neutral compound that forms a complex with a target ion. The ionophore is optically inactive in the visible spectrum and does not change absorbance or fluorescence depending on its state of complexation.

A chromoionophore is an ionophore that changes its optical properties in the visible spectrum depending on the state of complexation. Chromoionophores are preferably proton-sensitive dyes that change absorbance (and fluorescence in many cases) depending on its degree of hydrogen complexation (protonation). The chromionophores are preferably highly lipophilic to prevent them from leaching out of the optical ion-sensor matrix. Suitable chromionophores include Chromoionophore II and Chromionophore III. Chromionophore II exhibits light absorbance peaks at 520 nm and 660 nm and a fluorescent emission peak at 660 nm. Chromionophore III has light absorbance peaks at 500 nm and 650 nm and fluorescent emission peaks at 570 nm and 670 nm.

It should be stressed that the triggering ion released by the ionophore may be observed directly, as discussed above, or indirectly. For example, the intensity of the fluorescence signal may be a trigger for an event cascade within the cell. A large calcium current may cause the sensor to fluoresce brightly, for instance, whereas low calcium does not. The fluorescence may in turn excite a secondary dye in the particle that easily generates reactive oxygen species (ROS). The ROS would then attack the cell, effectively stimulating necrosis (cell death). Alternatively, instead of a secondary component within the particle, another particle may be added. This additional particle may comprise a photo-degradable polymer. When the primary sensory fluoresces, the emitted light will break apart the secondary particle, releasing its contents. The contents may, for example, be a drug that is therapeutic or cancer-fighting (stimulating apoptosis).

For optical ion sensors targeting cations, the additive can be any inert lipophilic component that has a negative charge associated with it. For optical ion sensors targeting anions, the additive is positively charged. The purpose of the additive is to embed charge sites within the polymer phase, and to help enforce charge neutrality within the optical ion sensor 112. The additive allows the polymer phase to carry an equal amount of charged particles as additive. The concentration ratio of additive to chromoionophore is preferably 1:1, thereby allowing the chromoionphore to become completely protonated or de-protonated. One suitable additive for optical ion sensors targeting negative ions is potassium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (KTFPB). The lipophilic, anionic component TFPB molecules are retained by the polymer phase, and the potassium ions are either complexed by the ionophore or expelled into the sample solution through diffusion. In one particular implementation, the optical ion sensor film is composed of a suspension produced from about 60 mg of DOS, 30 mg of PVC, and up to about 5 mg of additive, ionophore, and chromionophore.

Once the above components are dissolved into the polymer phase to form the optical ion sensor 112 and are exposed to a sample solution, the optical ion sensor 112 becomes active. It now continuously extracts or expels analyte cations (a system can work with anions as well using ion coextraction) depending on ion activity in the sample solution. With a 1:1 additive-chromoionophore ratio, and with zero target ions present in the sample solution, the optical ion sensor 112 remains completely protonated to achieve charge neutrality. As the target ion concentration increases, the ionophores in the optical ion sensor 112 extract the target ions into the optical ion sensor 112. To maintain charge neutrality of the optical ion sensor 112, hydrogen (hydronium) ions are stripped from the chromoionophores in the optical ion sensor 112 and expelled into the sample solution. The expelling of hydrogen ions alters the pH of the optical ion sensor 112, thereby altering its fluorescent properties. To detect analyte anions (for example, chloride or nitrite ions), the optical ion sensor uses ion-coextraction, as opposed to proton expulsion. To detect neutral analytes, an additional agent known to interact with the target analyte to yield an ion is added to the biological sample holder 104. An ionophore is then selected to detect the resultant ion.

The following is a non-limiting, illustrative list of target ion/ionophore pairings suitable for use in the optical ion sensors: potassium/Potassium Ionophore III (BME-44), sodium/Sodium Ionophore IV, sodium/Sodium Ionophore V, sodium/Sodium Ionophore VI, calcium/Calcium Ionophore III, and calcium/Calcium ionophore IV. For target anions, illustrative target ion/ionophore pairings include chloride/Chloride Ionophore III and nitrite/Nitrite Ionophore I.

The film of the optical ion sensor can be produced in various ways. In one implementation, as described above, a predetermined amount of the optical ion sensor suspension (i.e., the combined polymer phase, ionophore, additive, and chromionophore) is dissolved in a solvent, such as THF. The solution is then deposited, sprayed, or spun onto a surface. The solvent evaporates, leaving the optical ion sensor film on the surface.

In another implementation, the film is formed from a deposition of optical ion sensor microspheres. To produce the microspheres, an optical ion sensor emulsion is formed by injecting an optical ion sensor suspension dissolved in THF (e.g., 16 mL THF/100 mg PVC) into a pH buffered solution. The optical ion sensor suspension includes approximately 60 mg of DOS, 30 mg of PVC, and up to approximately 5 mg of chromionophore, additive, and ionophore. The emulsion is then submerged in a sonicating water bath. Typically, 50 μL of the optical ion sensor suspension/THF solution is injected into 1,000-1,500 μL of buffered solution. The resulting emulsion contains a mixture of spherical optical ion sensor particles ranging in size from 200 nm to 20 μm. The resulting emulsion can be spun, sprayed, or evaporated onto any surface to create a porous optical ion sensor membrane. Films formed from microspheres tend to expose a greater surface area of optical ion sensor to a given sample, yielding improved performance characteristics.

The biological sample holder 104 holds a biological sample for analysis by the cell assay system 100. The biological sample can include cells adhered to the walls of the biological sample holder 104, for example, in a monolayer, or cells suspended in a liquid buffer. The biological sample holder 104 is preferably transparent, or at least includes a transparent region through which the optical ion sensor 112 can be excited and through which the results of such excitement can be monitored.

The optical ion sensor 112 is illuminated with a light source 106 to excite the chromionophores suspended therein. The light source preferably can be tuned to generate one or more predetermined wavelengths of light, desirably in the visible portion of the electromagnetic spectrum, that are selected to excite the particular chromionophore used in the optical ion sensor 112. Alternatively, the light source may generate a wide spectrum light. In one implementation, the light source 106 is coupled to the optical ion sensor support 102.

The fluorescence of the optical ion sensor 112 is detected by a light sensor 108. The light sensor 108 may include a charge-coupled device, a fluorometer, a photomultiplier tube, or other suitable device for measuring fluorescence. In one implementation, a spectrophotofluorometer is used to satisfy the roles of the light source 106 and the light sensor 108. The light sensor 108 may also be coupled to the optical ion sensor support 102.

The optical ion sensor support 102 includes an agent introduction means 118. The agent introduction means 118 can include a pipette or an electro-mechanical dispenser device, such as a solenoid or electrostatically driven plunger or syringe.

The computing device 111 controls the various components of the cell assay system 100. The computing device 111 may be a single computing device or multiple computing devices providing the various functionalities used to control the cell assay system. These functionalities are provided by an excitation control module 126, an agent introduction module 130, and an analysis module 134. The excitation control module 126 controls the light source 108 to emit one or wavelengths of excitation light. The agent introduction module 130 controls the introduction of an agent into the biological sample holder 104 via an agent introduction means 118. The analysis module 134 analyzes the output of the light sensor 108, e.g., before and after an agent is introduced into the biological sample holder 104 to determine the effect of the agent on the cells in the biological sample holder 104. The analysis module 134 may also control the other modules in the computing device, i.e., the excitation control module 126 and the agent introduction module 130, to coordinate an assay protocol. The computing device 111 and/or devices may also include various user interface components, such as a keyboard, mouse, trackball, printer, and display.

A module may be implemented as a hardware circuit comprising custom VLSI circuits or gate arrays, off-the-shelf semiconductors such as logic chips, transistors, or other discrete components. A module may also be implemented in programmable hardware devices such as field programmable gate arrays, programmable array logic, programmable logic devices or the like.

Modules may also be implemented in software for execution by various types of processors. An identified module of executable code may, for instance, comprise one or more physical or logical blocks of computer instructions which may, for instance, be organized as an object, procedure, or function. Nevertheless, the executables of an identified module need not be physically located together, but may comprise disparate instructions stored in different locations which, when joined logically together, comprise the module and achieve the stated purpose for the module. A module of executable code may be a single instruction, or many instructions, and may even be distributed over several different code segments, among different programs, and across several memory devices.

The various modules are in communication with the various devices they control or obtain data from. They maybe connected over a local area network, wirelessly, over a bus, or over typical cables known in the art of computer interfaces for connecting computing devices with peripherals.

FIGS. 2A-2D depict various optical ion sensor arrangements suitable for use in various implementations of the cell assay system 100 of FIG. 1. FIG. 2A shows a first optical ion sensor arrangement that includes an optical ion sensor support 202 and a biological sample holder 204. The biological sample holder 204 includes a monolayer of cells 206 adhered to the biological sample holder 204. Alternatively, the biological sample holder 204 holds cells suspended in a buffer. The optical ion sensor support 202 and biological sample holder 204 correspond to the optical ion sensor support 102 and biological sample holder 104 of FIG. 1. An optical ion sensor film 210 is coupled to the distal end of the optical ion sensor support 202.

FIG. 2B illustrates an alternative optical ion sensor arrangement 250, which includes a biological sample holder 254 having therein a monolayer of cells 256 adhered to the surfaces of the biological sample holder 254, or cells suspended in a buffer. Instead of including an optical ion sensor film adhered to a support, the optical ion sensor arrangement 250 relies upon optical ion sensor particles 258 introduced into the cells 256 adhered to the biological sample holder 254.

To introduce optical ion sensors into cells, the optical ion sensors are produced as particles 258. The optical ion sensor particles 258 are fabricated in a fashion similar to the optical ion sensor film 112 described above. One such particle 258, the optical ion sensor nanosphere, is produced according to the following procedure. First a optical ion sensor suspension is dissolved in 500 μl of THF. The suspension preferably includes 60 mg of DOS, 30 mg of PVC and up to about 5 mg of chromoionophore, ionophore, and additive to form an optode solution. Then, 500 μl of CH2Cl2 is added to bring the total volume to 1 ml. Next, a PEG-lipid solution is prepared by dissolving a PEG-lipid (such as DSPE-PEG 550 or DSPE-PEG (2000) Folate) in 5 ml of a water, salt and buffer solution. A TAT peptide can be added to the PEG-lipid via an amine linkage to aid the resulting nanospheres in entering cells. Alternatively, it is possible to utilize a molecule that is not a PEG, e.g., a ganglioside attached to ceramide.

The nanospheres are formed by adding 100 μl of optode solution dropwise to 5 ml of the PEG-lipid solution while the solution is being sonicated by a probe tip sonicator. Additional sonication is performed for approximately 2-3 minutes. The resultant nanosphere solution is sprayed through a nitrogen-feed air gun into a beaker several times to remove excess solvent. If desired, the nanosphere solution is pushed through a 0.22 μm filter to remove the larger spheres.

The optical ion sensor particles 258 are introduced into the cells 256 in any suitable manner. In one method, the particles 258 are introduced into a buffer liquid deposited in the biological sample holder 254. A voltage source then generates a voltage sufficiently strong to electroporate the cells 256, thereby allowing the optical ion sensor particles 258 to enter directly into the cells. In another approach, the surfaces of the optical ion sensor particles 258 are first coated with a substance, for example transferrin or folate, which assist the particles 258 in crossing through cell membranes. The optical ion sensor particles 258 are introduced into a buffer in the biological sample holder 254, and the cells 256 bring the particles 258 into their interior in vesicles via endocytosis, pinocytosis, or phagocytosis, or similar biological processes. The substance applied to the optical ion sensor particles 258 breaks down the vesicle membrane, releasing the optical ion sensor particles 258 into the cell cytoplasm. In still other approaches, the particles 258 may be introduced into cells 256 using a glass needle or through ballistic bombardment.

In some embodiments, the sensor is attached to the exterior of a cell rather than introduced into the interior. If, for example, the activity of an ion channel is to be studied, the sensor may be attached to the cell proximate to the ion channel. This may be accomplished, for example, by raising antibodies against the ion-channel protein and, using conventional chemistries, covalently linking one or more such antibodies to a sensor particle as described above. The antibody-studded particles are released into a cell suspension and preferentially bind to the ion channel. This approach can be used to link ion-specific sensors to any feature on the exterior of the cell membrane to which antibodies can be raised. Alternatively, the sensors may be attached to the cell membrane by other suitable coupling chemistries, e.g., biotin-(strept)avidin complexing or polysaccharide binding. See the thesis “High Throughput Optical Sensor Arrays for Drug Screening” by Daniel I. Harjes (2006), available from the Massachusetts Institute of Technology and incorporated herein by reference.

FIG. 2C illustrates a second alternative optical ion sensor arrangement 270 that includes an optical ion sensor support 272 and a biological sample holder 274. An optical ion sensor film 276 is coupled to the distal end of the optical ion sensor support 272. A cell monolayer 278 adheres to the surfaces of the biological sample holder 274. Alternatively, cells are suspended in a buffer. In addition, optical ion sensor particles 280 are introduced into the cells of the cell monolayer 278. Preferably the chromionophores used in the optical ion sensor film 276 differ from the chromionophores used in the optical ion sensor particles 280. In particular, the different chromionophores desirably have distinguishable fluorescence characteristics such that an analysis module analyzing the output of a light sensor monitoring the optical ion sensor arrangement 270 can differentiate between the output of the optical ion sensor film 272 and the optical ion sensor particles 280. As a result, the analysis module can differentiate between intracellular target ion concentration and extracellular target ion concentration. In addition, the optical ion sensor film 272 may include ionophores different from those included in the optical ion sensor particles 280. Thus, the optical ion sensor arrangement 270 can monitor the concentrations of two different target ions.

FIG. 2D illustrates a third alternative optical ion sensor arrangement 290 that includes an electrode support 292 and a biological sample holder 294. The biological sample holder 294, in addition to a cell monolayer 296 or cells suspended in a buffer, includes a removable optical ion sensor film 298. The removable optical ion sensor film 298, for example, can be a glass cover slip or other transparent surface coated with an optical ion sensor film.

In still another embodiment, the optical ion sensor film is coated onto the inner surface of the biological sample holder. And in another approach, to accommodate the 96-well plate format often used in assays, one embodiment of the present invention utilizes round glass coverslips coated with the ion-exchange optode material along with the cells to be monitored. In this embodiment, each well contains a single sensor type to track a specific species of interest; the various sensor types may differ only in the ionophore employed and utilize the same or similar chromoionophores. The compound of interest is then added directly to the well. The 96-well plate is then placed in a standard plate-reader type fluorometer, and the fluorescence intensity is monitored with time.

In a typical implementation, a plurality of biological sample holders holding biological samples is provided. Biological samples introduced into the holders may include cells suspended in a buffer solution, but alternatively, cells may be adhered to the walls of the biological sample holders. Next, optical ion sensors are introduced into the biological sample holders as shown in FIGS. 2A and 2C, and/or are introduced into the cells themselves. Alternatively, the optical ion sensors can coat the walls of the biological sample holders. As described above, optical ion sensor particles can be introduced either by electroporating the cells via electrodes positioned in the biological sample holders or by the chemistry applied to the optical ion sensor particles breaching vesicle membranes within the cells. Similarly, the optical ion sensor sensors can be introduced into the cells using pico-injection, bead loading, a gene gun, or through liposomal delivery techniques known in the art. As described above, the optical ion sensors include at least one ionophore for selectively binding a predetermined ion. In some embodiments, this results in altering the pH of the optical ion sensor and a pH-sensitive chromionophore for optically indicating the concentration of the ion in a fluid surrounding the optical ion sensor. Ion concentration, in other words, is indicated by the pH of the optical ion sensor and the resulting fluorescence of the chromionophore.

An agent, such as a therapeutic, toxin, biological macromolecule (such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide), small molecule (of 2000 amu or less, 1000 amu or less or 500 amu or less), protein, virus, bacteria, chemical compound, mixture of chemical compounds, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues, or other biologically active agent may be introduced into one or more of the biological sample holders. In one particular implementation using an array of biological sample holders, no agent is introduced into a first row of biological sample holders to preserve a control. A first agent is introduced into a second row of biological sample holders. Additional agents are added to additional rows of the array of biological sample holders.

The fluorescence of the optical ion sensors introduced into the biological sample holders is monitored. The monitoring preferably begins prior to introduction of the agents and continues thereafter. Changes in ion concentration resulting from the introduced agents are then determined. By comparing the changes in ion concentration after adding an agent, one can determine the effect of the agent on the cells being tested.

FIGS. 3 and 4 present another embodiment 300 of a sensor in accord with the present invention. The sensor comprises a well 310 and micro-channels, indicated generally at 315, branching successively therefrom. The cells are placed in the well 310, sensor materials with different specificities line each of the channels 315, and a solution is flowed from the well 310 through the channels 315. When the fluorescence from the channels 315 is imaged, the pattern formed by changes in intensity may be visualized as an optical barcode, as depicted in FIG. 4, indicating the presence or absence of the various sensed materials in the solution.

Exemplary Applications: HERG Compatibility

The sensors of the present invention are suited to many different types of assays, or for the detection of compounds. In one exemplary application, a model cell system is used to screen for HERG compatibility. HERG (human ether-a-go-go) channels are implicated in the abnormal heart rhythms associated with Long QT Syndrome. It is believed that many compounds can block these channels and cause prolongation of the beat cycle of the heart. This abnormal rhythm can cause arrhythmia and can even lead to death. Accordingly, it is important that all drugs, regardless of therapeutic target, be screened for HERG compatibility.

With reference to FIG. 5, the model cell system to be used is Saccharomyces cerevisiae, as it contains a native ERG2 gene which is similar to the human HERG. It has been shown that known HERG inhibitors also interfere with the function of ERG2 in yeast. Much is known genetically about yeast, and gene knock-outs are commercially available, allowing for the screening of two types of yeast: the wild type (WT) containing the ERG2, and one mutated to knock-out the ERG2 gene.

With reference to FIG. 6, using both potassium and sodium sensors according to the present invention with both yeasts stimulated with a known HERG inhibitor, Haloperidol (an anti-psychotic drug), a change is observed in sodium and potassium upon stimulation of the cells with Haloperidol. In the ERG2 knock-out, there is no response. This example utilizes two cell types and two types of sensors, providing four different data signals, and the approach can be extended to an arbitrary number of sensors and cell types. Similarly, tissue can be used instead of culture.

Exemplary Applications: Ammonia Screening

As explained above, pH changes can be used to render neutral reaction products of interest measurable using an ion-exchange optode. For a sensor developed to monitor ammonia production in biological reactions (e.g., involving an enzyme such as transglutaminase (TGase)), for example, a low pH ionizes ammonia to ammonium, which may be extracted by a nonactin ionophore. This approach greatly expands the range of measurable species.

With reference to FIG. 7, an ammonia sensor is made by coating a 5 mm glass disk with an ammonium/ammonia-selective cocktail containing PVC, plasticizer, THF, potassium tetrakis (p-chlorophenyl)borate (KTpClPB), Chromoionophore III, and nonactin. A second, thin layer of 5% TEFLON is optionally applied to the coated disk to protect the ammonium cocktail from the high-salt environment required for the enzymatic reaction. These disks are placed in the bottom of individual wells in 96-well plates and the enzymatic reaction is set up to occur in the well, submerging the disks. Real-time monitoring of the reaction (through measuring the change in fluorescence of the optical sensor) is performed. Although these optodes are fairly large and coated with TEFLON, their response time is still approximately 2 minutes.

To utilize data monitoring in order to track the change in fluorescence of the sensors in the presence of an enzymatic reaction, a calibration curve of fluorescence readings of known concentrations of the ion of interest is first constructed. With reference to FIG. 8, this curve is typically sigmoidal, with the linear portion of the curve coinciding with the sensor's range of greatest accuracy. As depicted, in the case of a TGase reaction, the ammonia concentration currently susceptible to measurement is 10⁻⁴-10⁻³ M, although sensitivity is anticipated in the range of 10⁻⁵-10⁻³ M.

FIGS. 9 and 10 present the results of monitoring of the TGase reaction. Varying enzyme and substrate concentrations of the TGase reaction demonstrate the operation of the optical sensors. In particular, these results show that the higher the enzyme concentration or the higher the substrate concentration in the reaction, the faster ammonia is produced.

Further Applications

As discussed above, various embodiments of the present invention may be constructed to directly detect the presence of particular ions. As illustrated in Table 1 below, it is known to the art that certain diseases affect particular ion channels in a cell. Accordingly, assays for those ions utilizing the present invention may furnish a diagnostic tool to determine the presence of particular diseases. Accordingly, the scope of the present invention should be understood to also include the application of the heretofore-described subject matter to measure the ions set forth in the following tables, as well as their application to diagnose the presence of the associated diseases also appearing in the following tables.

Channel Gene Channel-forming unit/ligand OMIM Disease Cation channels: CHRNA1/ACHRA CHRNAI α, ACh 100690 Myasthenia congenita CHRNA4 CHRNA4 α, ACh 118504 Autosomal dominant nocturnal frontal lobe epilepsy CHRNB2 CHRNB2 β, ACh 118507 Autosomal dominant nocturnal frontal lobe epilepsy Polycystin-2 PKD2 α 173910 Autosomal dominant polycystic kidney disease (ADPKD) CNGA3 CNGA3 α, cGMP 60053 Achromatopsia 2 (color blindness) CNGB1 CNGB1 β, cGMP 600724 Autosomal recessive retinitis pigmentosa CNGB3 CNGB3 β, cGMP 605080 Achromatopsia 3 Sodium channels: Na.1.1 SCN1A α 182389 Generalized epilepsy with febrile seizures (GEFS+) Na.1.2 SCN2A α 182390 Generalized epilepsy with febrile and afebrile seizures) Na.1.4 SCN4A α 603967 Paramyotonia congenital, potassium aggressive myotonia, hyperkalemic periodic paralysis Na.1.5 SCN5a α 600163 Long-QT syndrome, progressive familial heart block type 1, Brugada syndrome (idiopathic ventricular arrhythmia) SCNIB SCN1B β 600235 Generalized epilepsy with febrile seizures (GEFS+) ENACα SCNNIA α 600228 Pseudohypoaldosteronism type 1 (PHA1) ENaCβ SCNN1B β 600760 PHA1, Liddle syndrome (dominant hypertension ENaCγ SCNN1G γ 600761 PHA1, Liddle syndrome Potassium channels: K, 1.1. KCNA1 α 176260 Episodic ataxia with myokymia KCNQI/K, LQT1 KCNQ1 α 192500 Autosomal dominant long-QT syndrome (Romano-Ward) Autosomal recessive long-QT syndrome with deafness (Jervell- Lange-Nielsen) KCNQ2 KCNQ2 α 602235 BFNC (epilepsy), also with myokymia KCNQ3 KCNQ3 α 602232 BFNC (epilepsy) KCNO4 KCNQ4 α 603537 DFNA2 (dominant hearing loss) HERG/KCNH2 KCNH2 α 152427 Long-QT syndrome Kir1.1/ROMK KCNJ1 α 600359 Bartter syndrome (renal salt loss, hypokalemic alkalosis) Kir2.1/IRK/KCNJ2 KCNJ2 α 600681 Long-QT syndrome with dysmorphic features (Andersen syndrome) Kir6.2/KATATP_(ATP) KCNJ11 α 600937 Persistent hyperinsulinemic hypoglycemia of infancy (PHHI) SURI SURI β 600509 PHHI KCNE1/Mink/ISK KCNE1 β 176261 Autosomal dominant long-QT syndrome (Romano-Ward) Autosomal recessive long-QT syndrome with deafness (Jervell- Lange-Nielson) KCNE2/MiRP1 KCNE2 β 603796 Long-QT syndrome KCNE3/MiRP2 KCNE3 β 604433 Periodic paralysis Calcium channels: Ca, 1.1 CACNA1S α 114208 Hypokalemic periodic paralysis, malignant hyperthermia Ca, 1.4 CACNA1F α 300110 X-linked congenital stationary night blindness Ca, 2.1 CACNA1A α 601011 Familial hemiplegic migraine, episodic staxia, spinocerebella ataxia type 6 RyRI RYR1 α 180901 Malignant hyperthermia, central core disease RyR2 RYR2 α 180902 Catecholaminergic polymorphic ventricular tachycardia, arrhythmogenic right ventricular dysplasia type 2 Chloride channels: CFTR ABCC7 α 602421 Cystic fibrosis, congenital bilateral asplasia of vas deference CIC-1 CLCN1 α 118425 Autosomal recessive (Becker) or dominant (Thomsen myotonia CIC-5 CLCN5 α 300008 Dent's disease (X-linked proteinuria and kidney stones) CIC-7 CLCN7 α 602727 Osteopetrosis (recessive or dominant) CIC-Kb CLCNKB α 602023 Bartter syndrome type III Barttin BSND β 606412 Bartter syndrome type IV (associated with sensorineural deafness) GLRA1 GLRA1 α, glycine 138491 Hyperekplexin (startle desease) GABAα1 GABRA1 α GABA 137160 Juvenile myoclonus epilepsy GABAγ2 GABRG2 γ, GABA 137164 Epilepsy Gap junction channels: Cx26 GJB2 121011 DFNB3 (autosomal dominant hearing loss) DFNB1 (autosomal recessive hearing loss) Cx30 GJB4 605425 DFNA3 Cx31 GJB3 603324 DFNA2 Cx32 GJB1 304040 CMTX (X-linked Charcot-Mari-Tooth neuropathy) AChR α7 Inflammation ClC7 Osteoporosis Ether-a-go-go (eag, erg, elk) Cancer Gardos channel Sickle cell anemia P2X7 Immune disorders TRPC6 Asthma, COPD TRPM1 Melanoma TRPM2 Asthma TRPM4 Immune disorders TRPM7 Stroke TRPM8 Prostate cancer TRPV1 Urinary incontinence, pain The third column classifies channel proteins into α, β, and γ subunits, where α subunits are always directly involved in pore formation, Several β subunits are only accessory (i.e., do not form pores), as is the case, for example, with SCN1B and barttin. Others (e.g. of ENaC and GABA receptors) participate in pore formation. For ligand-gated channels, the ligand is given. Note that GABA and glycine act from the extracellular side, whereas cGMP is an intracellular messenger.

Gene Accession ID Gene Locus Tissue Expression Sodium Channel Type/Disease SCN1A GDB: 118870 2q24 SCN1, vg type 1, α-subunit (280 KDa) Brain S71446 SCN1B GDB: 127281 19q13.1 Hs.89634, vg type 1 β₁ subunit Brain, heart, skeletal U12188-12104 (38 KDa) muscle L16242, L10338 SCN2A1 GDB: 120367 2q23 SCN2A, HBSCI, vg type II, α₁- Brain, peripheral nerve subunit (280(KDa) SCN2A2 CDB: 133727 2q23-24.1 HBSCH, vg type II, α₂-subunit vg Brain type II, β₂-subunit (33 KDa) SCN2B GDB: 118871 AF019498 SCN3A GDB: 132151 2q24-31 vg type III, α-subunit (280 kDa) Brain S69887 SCN4A GDB: 125181 17q23.1-25.3 SkM1, vg type IV α-subunit (260 kDa), Skeletal muscle L04216-L04236 hyperkalemic periodic paralysis, paramyotonia congentia, potassturn-aggravated myotonia SCN4B GDB: 125182 3q21 vg type IV, β-subunit, Heart, fetal skeletal SCN5A GDB: 132152 SkM2, hH1, vg type V, α-subunit, muscle long Q-T syndrome 3 SCN6A GDB: 132153 2q21-23 Hs99945, vg type VI, α-subunit Heart, uterus, fetal and denervated skeletal muscle SCN7A GDB: 228137 12q13 vg type VII, α-subunit Brain, spinal cord SCN8A GDB: 631695 vg type VIII, α-subunit, motor end-plate disease + ataxia in mice SCN9A GDB: 3750013 vg type IX, α-subunit Thyroid and adrenal neuroendocrine type gland SCN10A GDB: 750014 1pter-p36.3 hPN3, vg type X Sensory neurons, dorsal root ganglia SCNN1A GDB: 366596 12pt3 SCNN1, nvg type 1 α-subunit of Kidney, lung colon Z92978 ENaC SCNN1B GDB: 434471 16p12.2-p12.1 nvg 1 β-subunit, Liddle's syndrome, Kidney, lung colon pseudohypoaldosterontsm I SCNN1D GDB: 6053678 1p36.3-p36.2 DnaCh, nvg 1 δ-subunit Kidney, lung, colon SCNN1G GDB: 568769 16p122-p12.1 nvg 1 γ-subunit, Liddle's syndrome, Kidney, lung, colon X87160 pseudohypoaldosterontsm I U53835-53853 Calcium Channel Type/Disease CACNA1A GDB: 126432 19p13 P/Q type α_(1A-)subunit, eqisodic ataxia Brain (cortex, bulbus, CACNL1A4 Z80114-Z80155, 19p13.1 2, familial hemiplegic migraine, olfacorius, X99697, U79666 spinocerebellar ataxia 6; tottering, hippocarnpus, leaner, and rolling mice cerebellum, brain stem), motoneurons, kidney CACNA1B GDB: 580689 9q34 CACNN, N-type α_(1A-)subunit Central, peripheral CACNL1A5 M94172, M94173 nervous system CACNA1C GDB: 126094 12p13 CCHL1A1, L-type α_(1A-)subunit Heart, fibroblasts, lung, CACNL1A1 L29636, L29634, 12p13.3 smooth muscle (2 splice L29629 variants) CACNA1D GDB: 128872 3p14.3 CCHL1A2, L-type α_(1D)-subunit Brain, pancreas, CACNL1A2 3p21.3.2? neuroendocrine CACNA1E GDB: 434408 1q25-31 R-type α_(1C-)subunit Brain, skeletal muscle CACNL1A6 (end plate) CACNA1F GDB: 6053864 Xp11.23-11.22 α_(1F)-Subunit Retina CACNIAG AF27964 17q22 T-type α_(1G)-subunit Brain CACNA1S GDB: 126431 1q31-32 L-type α_(1B)-subunit (5% 212, 95% Skeletal muscle (brain, CACNL1A8 Z22672, L33798 190 kDa), malignant hyperthermia kidney) U30666-U30707 5, hypokalernic periodic paralysis CACNA2 GDB: 132010 7q21-22 CACNA2, CACNA2D1, α_(g8)-subunit α_(2A); skeletal muscle, CACNL2A Z28613, Z28609 (175 kDa), MHS3 heart, brain, ileum; α_(2B); Z28605, Z28602 brain; α_(2CVD); aorta Z28699, M76559 CACNB1 GDB: 132012 17q21-22 β₁-Subunit (524 aa, 54 kDa) β₁A/M; skeletal muscle CACNLB1 GDB: 1073281 β₁B/C; brain, heart, U86952-U86961 spleen M76560, L06111 GDB: 193328 CACNB2 GDB: 132014 10p12 MYSB, β₂-subunit β₂A/B/E; brain, heart, CACNLB2 Q08289 lung, aorta CACNB3 GDB: 341023 12q13 β₂-subunit (482 aa) Brain, heart, lung, spleen, CACNLB3 L27584 skeletal and smooth muscle, aorta, trachea, ovary, colon CACNB4 GDB: 6028693 2q22-23 β₂-subunit, lethargic mice Brain, kidney CACNG GDB: 132015 17q24 γ-Subunit (222 aa, 30 kDa) Skeletal muscle, lung CACNLG L07738 CACNG2 γ2-Subunit, stargazin, absence Brain epilepsy stargazer, waggler mice RYR1 GDB: 120359 19q13.1 Ryanodine receptor 1, Ca release Skeletal muscle, testis, channel, 3 splice variants, brain, submaxillary and malignant hyperthermia 1, central adrenal glands, spleen core disease RYR2 GDB: 125278 1pter-qter RYR2, calcium release channel Heart, smooth muscle 1q42.1-43 RYR3 GDB: 138451 15q14 RYR3, calcium release channel Brain, neonatal skeletal 15q14-15 muscle, adult diaphragm Potassium Channel Type/Disease KCNA1 GDB: 127903 12p13 RBK1, HUK1, MBK1, AEMK, Brain, nerve, heart, LO2750 Kv1.1, Shaker homolog 1, Shaker, skeletal muscle, retina, episodic ataxia 1 (with myokymia) pancreatic islet KCNA1B 3q26.1 Kvβ1.1, Kvβ1.3 (splice product), β-subunit KCNA2 GDB: 128062 12pter-qter HK4, Kv1.2, Shaker homolog 2 Brain, nerve, heart, X17622 pancreatic islet KCNA2B 1p36.3 Kvβ1.2, β-subunit KCNA8 GDB: 128079 1p13.3 Hs.1750, MK3, HLK3, HPCN3, Skeletal muscle, L23499 Kv1.3, Shaker homolog 3 lymphocytes (brain, lung, thymus, spleen) KCNA4 GDB: 126730 11p14 Hs.89647, Hs.1854, HK1, HPCN2, Brain, nerve, heart, fetal M60450 Kv1.4, Shaker homolog 4 skeletal muscle, M55514 pancreatic islet KCNA4L GDB: 386059 11q14 Shaker homolog type 4-like KCNA5 GDB: 127904 12p13.3-13.2 Hs.89509, HK2, HPCNI, Kv1.5 Brain, heart, kidney, lung, M83254 12p13 Shaker homolog 5 skeletal muscle, M60451 12p13.33-12.31 pancreatic islet KCNA6 GDB: 128080 12p13 HBK2, Kv1.6, Shaker homolog 6 Brain, pancreatic islet X17622 KCNA7 GDB: 127905 19q13.3 HAK6, Kv1.7 Shaker homolog 7 KCNA8 see KCNQ1 KCNA9 see KCNQ1 KCNA10 GDB: 5885822 Shaker homolog type 10, cGMP activated KCNB1 GDB: 128081 20q13.2 Kv2.1, Shab homolog 1 Brain, heart, kidney, retina, skeletal muscle KCNB2 Kv2.2, Shab homolog 2 Brain, heart, retina KCNC1 GDB: 128082 11p15.1 Kv3.1, Shaw homolog 1 Brain, skeletal muscle, S56770 spleen, lymphocytes M96747 KCNC2 GDB: 127906 19q13.3-13.4 Kv3.2, Shaw homolog 2 Brain KCNC3 GDB: 127907 19q13.3 Kv3.3, Shaw homolog 3 Brain, liver KCNC4 GDB: 127908 1p21 Kv3.4, HKSHIIIC, Shaw homolog 4 Brain, skeletal muscle KCND1 GDB: 128083 Kv4.1, Shal hormolog 1 Brain KCND2 GDB: 134771 RK5, Kv4.2, Shal homolog 2 Brain, heart, aorta KCND3 GDB: 134772 Kv4.3, KSHIVB, Shal homolog 3 KCNE1 GDB: 127909 21q22.1-22.2 MinK, ISK, vg Isk homolog 1 (129 Kidney, submandibular aa), long Q-T syndrome 5 gland, uterus, heart, cochlea, retina KCNMA1 GDB: 386031 10pter-qter SLO, Hs.62679, α-subunit member Fetal skeletal muscle U09383-4 7q32.1 1, α-subunit of maxiK or BK U02632 channel KCNMB1 GDB: 6099615 5q34 hSLO-β, β-subunit member 1 (191 Smooth, fetal skeletal U42600 aa), β-subunit of max1K or BK muscle, brain channel (hippocampus, corpus callosum) KCNN1 U69883 SK(Ca)1, small-conductance Ca- Brain, heart activated K channel, apamin- insensitive KCNN2 SK(Ca)2, apamin sensitive Brain, adrenal gland KCNN3 Y08263 1q? SK(Ca)3, small-conductance Ca- Brain, heart, (human AA285078 activated K channel, intermediate embryonic) skeletal apamin sensitivity muscle, liver KCNN4 AF022150 19q13.2 IK1, intermediate-conductance T lymphocytes, colon, AF022797 Ca-activated K channel, KCa4, smooth muscles, AF033021 SK4, Gantos channel prostata, red blood cells, AF000972 neurons KCNQ1 GDB: 741244 11p15.5 KCNA9, (KV)LQT1, KQT-like Heart, cochlea, kidney, U40990 subfamily member 1, long Q-T lung, placenta, colon syndrome 1 KCNQ2 GDB: 9787229, 20q13.3 KQT-like subfamily member 2 (872 Brain Y15065, aa) AF033348 KCNQ3 GDB: 9787230 8q24.22-24.3 KQT-like subfamily member 3 (825 Brain AF033347 aa) HERG GDB: 407638 7q35-36 HERG, similar to ether-a-go go Brain, heart (eag), IKr, long Q-T syndrome 2 KCNJ1 GDB: 204206 11q24 ROMK1, Kirl.1, Hs.463, Kidney, pancreatic islets U65406, U12541 Bartter/hyperprostaglandin E syndrome KCNJ2 GDB: 278964 17pter-qter IRK1, Kir2.1, Hs.1547 Muscle, neural tissue, U12507 heart KCNJ3 GDB: 278325 2q24.1 GIRK1, Kir3.1 Heart, cerebellum U50964 KCNJ4 GDB: 374080 22q13.1 HIR, HIRK1, HIRK2, Kir2.3 Heart, skeletal muscle, Z97056 brain KCNJ5 GDB: 547948 11q24 CIR, KATP1, GIRK4, Kir3.4 Heart, pancreas KCNJ6 GDB: 547949 21q22.1 KCNJ7, GIRK2, KATP2, BIR1, Cerebellum, pancreatic U24660 Kir3.2, ataxia, weaver mice islet KCNJ8 GDB: 633096 12p11.23] Kir6.1, uKATP, ubiquitious K_(ATP) Brain, heart, skeletal, α-subunit smooth muscle, others KCNJ10 GDB: 3750203 1q22-23] Kir1.2, Kir4.1 Glia KCNJ11 GDB: 7009893 [11p15.1] Kir6.2, BIR, K(ATP) α-subunit, Pancreatic islets hyperinsulinemic hypoglycemia KCNJ12 GDB: 4583927 [17p11.1] Kir2.2 KCNJ15 GDB: 6275865 [21q22.2] Kir4.2 KCNJN1 GDB: 6108062 [ ] Kir2.2v, subfamily inhibitor 1 SUR1 GDB: 591970 [11p15.1] SUR(1), sulfonylurea receptor, Pancreatic islets K(ATP) β-subunit, hyperinsulinemic hypoglycemia SUR2 12p12.1] SUR2, SUR2A, B, sulfonylurea 2A: heart, 2B: brain, liver, receptor 2 (1545-aa), β-subunit of skeletal, smooth muscle, K(ATP) urinary bladder KCNK1 GDB: 6045446 1q42-43 DPK, TWIK1 Kidney KCNK2 1q41 TREK1 Brain KCNK3 GDB: 9773281 2p23 TASK Kidney

Therapeutic Target Enzyme Family Assay Alzheimer's CMGC ERK2 (P42mapk) Alzheimer's Phospholipase PLA2 Alzheimer's Cyclooxygenases COX2 Alzheimer's CaMK MARK1 Alzheimer's CaMK MARK2 Alzheimer's AGC PKCalpha Alzheimer's AGC PKCgamma Alzheimer's AGC PKCgamma Alzheimer's Cysteine proteases caspase-3 Alzheimer's Cysteine proteases caspase-6 Alzheimer's Aspartic proteases BACE-1 (beta-secretase) Alzheimer's Aspartic proteases cathepsin D Alzheimer's Aspartic proteases cathepsin F Alzheimer's Metalloproteases ACE Alzheimer's Metalloproteases ACE Alzheimer's Metalloproteases TACE Alzheimer's NO synthases constitutive NOS (cerebellar) Alzheimer's Monoamine &neurotransmitter acetylcholinesterase synthesis & metabolism Alzheimer's Monoamine &neurotransmitter COMT (catechol-O-methyl transferase) synthesis & metabolism Alzheimer's Monoamine &neurotransmitter MAO-A synthesis & metabolism Alzheimer's Monoamine &neurotransmitter MAO-B synthesis & metabolism Alzheimer's Monoamine &neurotransmitter tyrosine hydroxylase synthesis & metabolism Alzheimer's Phospholipase C PLC Alzheimer's Miscellaneous enzymes xanthine oxidase/superoxide O2-scavenging Dependence/Addiction AGC PKA Dependence/Addiction AGC PKCalpha Dependence/Addiction AGC PKCbeta 1 Dependence/Addiction AGC PKCbeta 2 Dependence/Addiction AGC PKCdelta Dependence/Addiction Monoamine &neurotransmitter GABA transaminase synthesis & metabolism Dependence/Addiction Cyclases adenylyl cyclase (stimulated) Dependence/Addiction Phospholipase C PLC Dependence/Addiction ATPase ATPase (Na+/K+) Inflammation/Arthritis/Allergy RTK EGFR kinase Inflammation/Arthritis/Allergy RTK FLT-1 kinase (VEGFR1) Inflammation/Arthritis/Allergy RTK KDR kinase (VEGFR2) Inflammation/Arthritis/Allergy CTK Fyn kinase Inflammation/Arthritis/Allergy CTK HCK Inflammation/Arthritis/Allergy CTK Lck kinase Inflammation/Arthritis/Allergy CTK Lyn kinase Inflammation/Arthritis/Allergy CTK ZAP70 kinase Inflammation/Arthritis/Allergy CMGC ERK2 (P42mapk) Inflammation/Arthritis/Allergy CMGC JNK 1 Inflammation/Arthritis/Allergy CMGC JNK 2 Inflammation/Arthritis/Allergy CMGC P38alpha kinase Inflammation/Arthritis/Allergy Phospholipase PLA2 Inflammation/Arthritis/Allergy Cyclooxygenases COX1 Inflammation/Arthritis/Allergy Cyclooxygenases COX2 Inflammation/Arthritis/Allergy TXA2 synthetase TXA2 synthetase Inflammation/Arthritis/Allergy CaMK MAPKAPK2 Inflammation/Arthritis/Allergy AGC PKA Inflammation/Arthritis/Allergy Lipoxygenases 12-lipoxygenase Inflammation/Arthritis/Allergy Lipoxygenases 15-lipoxygenase Inflammation/Arthritis/Allergy Serine proteases elastase Inflammation/Arthritis/Allergy Serine proteases cathepsin G Inflammation/Arthritis/Allergy Serine proteases kallikrein Inflammation/Arthritis/Allergy Serine proteases tryptase Inflammation/Arthritis/Allergy Cysteine proteases caspase-1 Inflammation/Arthritis/Allergy Cysteine proteases caspase-4 Inflammation/Arthritis/Allergy Cysteine proteases caspase-5 Inflammation/Arthritis/Allergy Cysteine proteases cathepsin B Inflammation/Arthritis/Allergy Cysteine proteases cathepsin X Inflammation/Arthritis/Allergy Aspartic proteases cathepsin F Inflammation/Arthritis/Allergy Metalloproteases MMP-1 Inflammation/Arthritis/Allergy Metalloproteases MMP-2 Inflammation/Arthritis/Allergy Metalloproteases MMP-3 Inflammation/Arthritis/Allergy Metalloproteases MMP-7 Inflammation/Arthritis/Allergy Metalloproteases MMP-8 Inflammation/Arthritis/Allergy Metalloproteases MMP-9 Inflammation/Arthritis/Allergy Metalloproteases MMP-13 Inflammation/Arthritis/Allergy Metalloproteases MT1-MMP (MMP-14) Inflammation/Arthritis/Allergy Metalloproteases TACE Inflammation/Arthritis/Allergy Phosphatases phosphatase CD45 Inflammation/Arthritis/Allergy Phosphodiesterases PDE2 Inflammation/Arthritis/Allergy Phosphodiesterases PDE4 Inflammation/Arthritis/Allergy Phosphodiesterases acid sphingomyelinase Inflammation/Arthritis/Allergy Monoamine & neurotransmitter HNMT (histamine N-methyltransferase) synthesis & metabolism Inflammation/Arthritis/Allergy Miscellaneous enzymes myeloperoxidase Inflammation/Arthritis/Allergy Miscellaneous enzymes xanthine oxidase/superoxide O2-scavenging Neuroprotection RTK TRKB Neuroprotection CMGC CDK5 Neuroprotection CMGC DYRK1a Neuroprotection CMGC ERK1 Neuroprotection CMGC ERK2 (P42mapk) Neuroprotection MCGC JCK 3 Neuroprotection Cyclooxygenases COX1 Neuroprotection Cyclooxygenases COX2 Neuroprotection CaMK CaMK2alpha Neuroprotection AGC PKA Neuroprotection Cysteine proteases caspase-3 Neuroprotection Phosphodiesterases PDE1 Neuroprotection Phosphodiesterases PDE6 Neuroprotection NO synthases constitutive NOS (endothelial) Neuroprotection NO synthases constitutive NOS (cerebellar) Neuroprotection Monoamine & neurotransmitter acetylcholinesterase syntheses & metabolism Neuroprotection Monoamine & neurotransmitter COMT (catechol-O-methyl transferase) syntheses & metabolism Neuroprotection Monoamine & neurotransmitter GABA transaminase syntheses & metabolism Neuroprotection Monoamine & neurotransmitter HNMT (histamine N-methyltransferase) syntheses & metabolism Neuroprotection Monoamine & neurotransmitter MAO-A syntheses & metabolism Neuroprotection Monoamine & neurotransmitter MAO-A syntheses & metabolism Neuroprotection Monoamine & neurotransmitter PNMT (phenylethanoiamine-N-methyl syntheses & metabolism transferase) Neuroprotection Monoamine & neurotransmitter tyrosine hydroxylase syntheses & metabolism Neuroprotection Cyclases guanylyl cyclase (basal) Neuroprotection Cyclases guanylyl cyclase (stimulated) Neuroprotection ATPase ATPase (Na+/K+) Neuroprotection Miscellaneous enzymes xanthine oxidase/superoxide O2-scavenging Parkinson CMGC JNK 1 Parkinson Phospholipase PLA2 Parkinson Cyclooxygenases COX2 Parkinson Cysteine proteases caspase-3 Parkinson NO synthases constitutive NOS (cerebellar) Parkinson Monoamine & neurotransmitter acetylcholinesterase syntheses & metabolism Parkinson Monoamine & neurotransmitter COMT (catechol-O-methyl transferase syntheses & metabolism Parkinson Monoamine & neurotransmitter MAO-A syntheses & metabolism Parkinson Monoamine & neurotransmitter MAO-B syntheses & metabolism Cancer RTK Axl kinase Cancer RTK c-kit kinase Cancer RTK c-kit kinase Cancer RTK EGFR kinase Cancer RTK EphA1 kinase Cancer RTK EphA3 kinase Cancer RTK EphA4 kinase Cancer RTK EphB2 kinase Cancer RTK FGFR1 kinase Cancer RTK FGFR2 kinase Cancer RTK FGFR3 kinase Cancer RTK FGFR4 kinase Cancer RTK FLT-1 kinase (VEGFR1) Cancer RTK FLT-3 kinase Cancer RTK FLT-4 kinase (VEGFR3) Cancer RTK Fms/CSFR kinase Cancer RTK HER2/ErbB2 kinase Cancer RTK HER4/ErbB4 kinase Cancer RTK KDR kinase (VEGFR2) Cancer RTK PDGFRalpha kinase Cancer RTK PDGFRbeta kinase Cancer RTK Ret kinase Cancer RTK TIE2 kinase Cancer RTK TRKA Cancer CTK Abl kinase Cancer CTK BLK Cancer CTK BMX (Bk) kinase Cancer CTK BRK Cancer CTK BTK Cancer CTK CSK Cancer CTK FAK Cancer CTK Fes kinase Cancer CTK Fyn kinase Cancer CTK JAK2 Cancer CTK JAK3 Cancer CTK Lck kinase Cancer CTK PYK2 Cancer CTK Src kinase Cancer CTK Syk Cancer CTK Yes kinase Cancer CMGC CDC2/CDK1 (cycB) Cancer CMGC CDK2 (cycE) Cancer CMGC CDK4 (cycD1) Cancer CMGC CDK5 Cancer CMGC CK2 (casein kinase 2) Cancer CMGC DYRKla Cancer CMGC ERK1 Cancer CMGC ERK2 (P42mapk) Cancer CMGC HIPK2 Cancer CMGC IKKalpha Cancer CMGC IKKbeta Cancer CMGC JNK 1 Cancer CMGC JNK 2 Cancer CMGC NEK1 Cancer CMGC NEK2 Cancer CMGC NEK4 Cancer CMGC p38alpha kinase Cancer CMGC p38beta 2 kinase (SAPK2b2) Cancer CMGC p38delta kinase Cancer CMGC p38ganuna kinase Cancer Cyclooxygenases COX2 Cancer CaMK CaMK1delta Cancer CaMK CaMK Cancer CaMK CHK1 Cancer CaMK CHK2 Cancer CaMK DAPK1 Cancer CaMK DAPK2 Cancer CaMK MAPKAPK2 Cancer CaMK MAPKAPK3 Cancer CaMK MAPKAPK5 (PRAK0 Cancer CaMK MAARK1 Cancer CaMK MARK2 Cancer CaMK MARK4 Cancer CaMK Pim 1 kinase Cancer CaMK Pirn2 kinase Cancer AGC Akt1/PKBalpha Cancer AGC Akt2/PKBbeta Cancer AGC Akt3/PKBgamma Cancer AGC AurA/Aur2 kinase Cancer AGC AurB/Aur1 kinase Cancer AGC AurC/Aur3 kinase Cancer AGC P70S6Ke Cancer AGC PDK1 Cancer AGC PKA Cancer AGC PKCalpha Cancer AGC PKCbeta 1 Cancer AGC PKCbeta 2 Cancer AGC PKCdelta Cancer AGC PKCgamma Cancer AGC PKG2 Cancer AGC ROCK1 Cancer AGC ROCK2 Cancer AGC RSK2 Cancer AGC SGK1 Cancer Lipoxygenases 12-lipoxygenase Cancer TKL RAF-1 kinase Cancer STE MEK1/MAP2K1 Cancer STE MKK4/JNK1 Cancer STE MKK6 Cancer STE PAK1 Cancer STE PAK2 Cancer Serine proteases elastase Cancer Serine proteases cathepsin G Cancer Cysteine proteases caspase-2 Cancer Cysteine proteases caspase-3 Cancer Cysteine proteases caspase-8 Cancer Cysteine proteases caspase-9 Cancer Cysteine proteases cathepin B Cancer Cysteine proteases cathepsin H Cancer Cysteine proteases cathepsin L Cancer Cysteine proteases cathepsin X Cancer Aspartic proteases cathepsin D Cancer Aspartic proteases cathepsin E Cancer Metalloproteases MMP-1 Cancer Metalloproteases MMP-2 Cancer Metalloproteases MMP-3 Cancer Metalloproteases MMP-7 Cancer Metalloproteases MMP-8 Cancer Metalloproteases MMP-9 Cancer Metalloproteases MMP-12 Cancer Metalloproteases MMP-13 Cancer Metalloproteases MT1-MMP (MMP-14) Cancer Metalloproteases TACE Cancer' Metalloproteases MMP-1 Cancer Phosphatases phosphatase 1B Cancer Phosphatases phosphatase 2B Cancer Phosphodiesterases PDE2 Cancer Phosphodiesterases PDE4 Cancer Phosphodiesterases PDE5 Cancer Phosphodiesterases acid spingomyelinase Cancer NO synthases constitutive NOS (endothelial) Cancer NO synthases constitutive NOS (cerebellar) Cancer Cyclases adenylyl cyclase (basal) Cancer Cyclases adenylyl cyclase (stimulated) Cancer Phospholipase C PLC Cancer Miscellaneous enzymes myeloperoxidase Cancer Miscellaneous enzymes xanthine oxidase/superoxide O2-scavenging Diabetes RTK Ax1 kinase Diabetes RTK EGFR kinase Diabetes RTK IGFIR kinase Diabetes CMGC ERK2 (P42mapk) Diabetes CMGC Jnk1 Diabetes Cyclooxygenases COX2 Diabetes TXA2 synthetase TXA2 synthetase Diabetes CaMK AMPKalpha Diabetes AGC Akt1/PKBalpha Diabetes AGC Akt2/PKBbeta Diabetes AGC Akt3/PKBgamma Diabetes AGC PDK1 Diabetes AGC PKA Diabetes AGC PKCalpha Diabetes AGC PKCbeta 1 Diabetes AGC PKCbeta 2 Diabetes AGC PKCgamma Diabetes AGC SGK2 Diabetes Metalloproteases ACE Diabetes Metalloproteases MMP-1 Diabetes Metalloproteases MMP-2 Diabetes Metalloproteases MMP-3 Diabetes Metalloproteases MMP-7 Diabetes Metalloproteases MMP-8 Diabetes Metalloproteases MMP-9 Diabetes Metalloproteases MT1-MMP (MMP-14) Diabetes Metalloproteases TACE Diabetes Phosphodiesterases PDE3 Diabetes Phosphodiesterases PDE4 Diabetes Phosphodiesterases PDE5 Diabetes NO synthases constitutive NOS (endothelial) Diabetes Monoamine & neurotransmitter acetylcholinesterase synthesis & metabolism Diabetes Monoamine & neurotransmitter GABA transaminase synthesis & metabolism Diabetes Monoamine & neurotransmitter MAO-B synthesis & metabolism Diabetes Cyclases adenylyl cyclase (basal) Diabetes Miscellaneous enzymes acetylCoA synthetase Diabetes Miscellaneous enzymes HMG-CoA reductase Diabetes Miscellaneous enzymes xanthine oxidase/superoxide O2-scavenging Metabolic Diseases Cyclooxygenases COX2 Metabolic Diseases AGC PKA Metabolic Diseases Metalloproteases ACE Metabolic Diseases Phosphodiesterases PDE3 Metabolic Diseases Phosphodiesterases PDE4 Metabolic Diseases NO synthases constitutive NOS (endothelial) Metabolic Diseases Miscellaneous enzymes acetylCoA synthetase Metabolic Diseases Miscellaneous enzymes HMG-CoA reductase Metabolic Diseases Miscellaneous enzymes xanthine oxidase/superoxide 02-scavenging Obesity CTK PYK2 Obesity CMGC JNK1 Obesity CaMK AMPJakoga Obesity AGC PKA Obesity Metalloproteases ACE Obesity Metalloproteases ACE Obesity Phosphatases phosphatase 1B Obesity Phosphodiesterases PDE2 Obesity Phosphodiesterases PDE3 Obesity Monoamine & neurotransmitter acetylcholinesterase synthesis & metabolism Obesity ATPase ATPase (Na+/K+) Obesity Miscellaneous enzymes HMG-CoA reductase Reproduction Phospholipase PLA2 Reproduction Cyclooxygenases COX1 Reproduction Cyclooxygenases COX2 Reproduction Phosphodiesterases PDE5 Reproduction NO synthases constitutive NOS (endothelial) Reproduction Cyclases guanylyl cyclase (stimulated) Cystic Fibrosis Phospholipase PLA2 Cystic Fibrosis TXA2 synthetase TXA2 synthetase Cystic Fibrosis AGC PKA Cystic Fibrosis AGC PKCbeta 1 Cystic Fibrosis AGC PKCbeta 2 Cystic Fibrosis Serine proteases elastase Cystic Fibrosis Serine proteases cathepsin G Cystic Fibrosis Metalloproteases MMP-2 Cystic Fibrosis Phosphodiesterases PDE3 Cystic Fibrosis Phosphodiesterases PDE5 Cystic Fibrosis Cyclases adenylyl cyclase (stimulated) Cystic Fibrosis Phospholipase C PLC Cystic Fibrosis Miscellaneous enzymes myeloperoxidase Immunosuppression Profile RTK EGFR kinase Immunosuppression Profile CTK JAK3 Immunosuppression Profile CMGC ERK2 (P42mapk) Immunosuppression Profile Cyclooxygenases COX1 Immunosuppression Profile Cyclooxygenases COX2 Immunosuppression Profile Serine proteases elastase Immunosuppression Profile Serine proteases cathepsin G Immunosuppression Profile Serine proteases tryptase Immunosuppression Profile Cysteine proteases cathepsin B Immunosuppression Profile Metalloproteases ECE-1 Immunosuppression Profile Metalloproteases ECE-1 Immunosuppression Profile Metalloproteases MMP-1 Immunosuppression Profile Metalloproteases MMP-2 Immunosuppression Profile Metalloproteases MMP-9 Immunosuppression Profile Phosphatases phosphatase CD45 Immunosuppression Profile Phosphodiesterases PDE4 Immunosuppression Profile Phosphodiesterases acid spingomyelinase Immunosuppression Profile Cyclases adenylyl cyclase (basal) Immunosuppression Profile Cyclases adenylyl cyclase (stimulated) Migraine Cyclooxygenases COX2 Migraine NO synthases constitutive NOS (cerebellar) Migraine Monoamine & neurotransmitter GABA transaminase synthesis & metabolism Migraine Cyclases guanylyl cyclase (stimulated) Pain CMGC ERK2 (42mapk) Pain Phospholipase PLA2 Pain Cyclooxygenases COX1 Pain Cyclooxygenases COX2 Pain AGC PKA Pain Serine proteases elastase Pain Metalloproteases MMP-1 Pain Metalloproteases MMP-2 Pain Metalloproteases MMP-3 Pain Metalloproteases MMP-7 Pain Phosphodiesterases PDE4 Pain NO synthases constitutive NOS (endothelial) Pain NO synthases constitutive NOS (cerebellar) Pain Monoamine & neurotransmitter GABA transaminase synthesis & metabolism Pain Monoamine & neurotransmitter MAO-A synthesis & metabolism Pain Monoamine & neurotransmitter MAO-B synthesis & metabolism Pain Monoamine & neurotransmitter tyrosine hydroxylase synthesis & metabolism Pain Miscellaneous enzymes xanthine oxidase/superoxide 02-scavenging

Thus, according to various features, the systems, methods and devices of the invention provide, without limitation, a novel approach for using optodes and cells for measurement, including drug discovery and the monitoring of enzymatic reactions; the coating of an ion-selective PVC cocktail with TEFLON to prevent salt interference; an approach for using the optodes for extracellular measurement in a 96-well plate format, and for HERG and ammonia screening. The invention also provides a microdevice for combining optodes with cell measurements, sensor inserts for a well plate (such as PVC-coated glass disks), and bio-compatible coatings for optodes. Commercial applications for the invention include, without limitation, drug discovery, clinical monitoring, toxicity studies, chemical and biological detection, and the like.

Additional applications and protocols for analyzing ion-channel activity are described in U.S. Pat. No. 6,969,449, the entirety of which is incorporated by reference. Such protocols can be readily adapted for use with the optical ion sensors and cell assay systems described herein.

It will therefore be seen that the foregoing represents a highly advantageous approach to measurements using optical biosensor arrays. The terms and expressions employed herein are used as terms of description and not of limitation and there is no intention, in the use of such terms and expressions, of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. 

1. A method for measuring a chemical reaction occurring within a living cell, the method comprising: (a) introducing at least one sensor into the cell without compromising the viability of the cell, the at least one sensor producing an observable signal indicating a value of a parameter associated with the reaction; wherein the at least one sensor is a nanosphere containing a PEG-lipid coating; and (b) using the signal to ascertain the parameter value.
 2. The method of claim 1 wherein the parameter value corresponds to the concentration of a product of the reaction.
 3. The method of claim 1 further comprising the step of stimulating the cell so as to affect the reaction.
 4. The method of claim 3 wherein the cell is stimulated by at least one of a compound, a pathogen, a pharmaceutical compound, or a potential toxin.
 5. The method of claim 1 further comprising the step of placing at least one sensor in proximity to the cell.
 6. The method of claim 5 wherein the at least one proximate sensor is covalently attached to the exterior membrane of the cell.
 7. The method of claim 5 wherein the at least one proximate sensor is attached to the cell membrane proximate to an ion channel of the cell.
 8. The method of claim 7 wherein the at least one proximate sensor is attached to the cell membrane via an antibody specific to the ion channel.
 9. The method of claim 1 wherein the at least one sensor comprises an ion-selective optode.
 10. The method of claim 9 wherein the reaction produces an ionic product.
 11. The method of claim 9 wherein the reaction produces a non-ionic product, and further comprising the step of ionizing the product to facilitate monitoring using the optode.
 12. The method of claim 9 wherein (i) the optode comprises an ion-selective ionophore, a source of triggering ions, and a signaling agent responsive to the triggering ions, (ii) the ionphore is selective for the ionic reaction product, and (iii) upon binding of a reaction-product ion, a triggering ion is released to interact with the signaling agent and thereby produce the signal.
 13. The method of claim 12 wherein the signal has a strength indicative of the concentration of the triggering ion, which is itself indicative of the concentration of the ionic reaction product.
 14. The method of claim 1 wherein monitoring the signal comprises observing the optical activity of the at least one sensor.
 15. The method of claim 14 wherein the signal comprises fluorescence.
 16. The method of claim 15 wherein monitoring the response comprises observing the optical activity of the at least one sensor using a fluorometer.
 17. The method of claim 10 wherein a first of the sensors and a second of the sensors each respond to different ions.
 18. A method for measuring a chemical reaction occurring within a living cell, the method comprising: (a) attaching at least one sensor to the exterior membrane of the cell without compromising the viability of the cell or significantly affecting the chemical reaction being monitored, the sensor producing an observable signal indicating a value of a parameter associated with the reaction; and (b) using the signal to ascertain the parameter value.
 19. The method of claim 18 wherein the parameter value corresponds to the concentration of a product of the reaction.
 20. The method of claim 18 further comprising the step of stimulating the cell so as to affect the reaction.
 21. The method of claim 18 wherein the cell is stimulated by at least one of a compound, a pathogen, a pharmaceutical compound, or a potential toxin.
 22. The method of claim 18 wherein the at least one sensor is attached to the cell membrane proximate to an ion channel of the cell.
 23. The method of claim 22 wherein the at least one sensor is attached to the cell membrane via an antibody specific to the ion channel.
 24. The method of claim 18 wherein the at least one sensor comprises an ion-selective optode.
 25. The method of claim 24 wherein the reaction produces an ionic product.
 26. The method of claim 24 wherein the reaction produces a non-ionic product, and further comprising the step of ionizing the product to facilitate monitoring using the optode.
 27. The method of claim 24 wherein (i) the optode comprises an ion-selective ionophore, a source of triggering ions, and a signaling agent responsive to the triggering ions, (ii) the ionphore is selective for the ionic reaction product, and (iii) upon binding of a reaction-product ion, a triggering ion is released to interact with the signaling agent and thereby produce the signal.
 28. The method of claim 27 wherein the signal has a strength indicative of the concentration of the triggering ion, which is itself indicative of the concentration of the ionic reaction product.
 29. The method of claim 18 wherein monitoring the signal comprises observing the optical activity of the at least one sensor.
 30. The method of claim 29 wherein the signal comprises fluorescence.
 31. The method of claim 30 wherein monitoring the response comprises observing the optical activity of the at least one sensor using a fluorometer.
 32. The method of claim 18 wherein a first of the sensors and a second of the sensors each respond to different ions.
 33. The method of claim 18 further comprising the step of introducing at least one internal sensor into the cell without compromising the viability of the cell, the at least one sensor emitting an observable signal indicating a value of a parameter associated with the reaction. 34-44. (canceled)
 45. The method of claim 18, wherein the at least one sensor is a nanosphere containing a PEG-lipid coating.
 46. The method of claim 1, wherein the least one sensor comprises an ionophore, a chromoionophore, a polymer, and an additive.
 47. The method of claim 46, wherein the ionophore is Potassium Ionophore III, Sodium Ionophore IV, Sodium Ionophore V, Sodium Ionophore VI, Calcium Ionophore III, or Calcium Ionophore IV.
 48. The method of claim 46, wherein the chromoionophore is Chromoionophore II or Chromoionophore III.
 49. The method of claim 46, wherein the polymer is polyvinyl chloride.
 50. The method of claim 46, wherein the least one sensor further comprises a plasticizer.
 51. The method of claim 50, wherein the plasticizer is dioctyl sebacate.
 52. The method of claim 1, wherein the at least one sensor comprises an ionophore, a chromoionophore, polyvinyl chloride, dioctyl sebacate, and an additive.
 53. The method of claim 52, wherein the chromoionophore is Chromoionophore III.
 54. The method of claim 1, wherein the PEG-lipid is DSPE-PEG 500 or DSPE-PEG (2000) Folate.
 55. The method of claim 46, wherein the PEG-lipid is DSPE-PEG 500 or DSPE-PEG (2000) Folate. 